ARTICLE https://doi.org/10.1038/s41467-021-24097-6 OPEN TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells ✉ Bruno Silva 1,4, Rajika Arora 1,3,4, Silvia Bione 2 & Claus M. Azzalin 1 Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. While the notion that 1234567890():,; spontaneous DNA damage at telomeres is required to initiate ALT, the molecular triggers of this physiological telomere instability are largely unknown. We previously proposed that the telomeric long noncoding RNA TERRA may represent one such trigger; however, given the lack of tools to suppress TERRA transcription in cells, our hypothesis remained speculative. We have developed Transcription Activator-Like Effectors able to rapidly inhibit TERRA transcription from multiple chromosome ends in an ALT cell line. TERRA transcription inhi- bition decreases marks of DNA replication stress and DNA damage at telomeres and impairs ALT activity and telomere length maintenance. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby triggering BIR and promoting telomere elongation. Our data point to TERRA transcription manipulation as a potentially useful target for therapy. 1 Instituto de Medicina Molecular João Lobo Antunes (iMM), Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugal. 2 Computational Biology Unit, Institute of Molecular Genetics Luigi Luca Cavalli-Sforza, National Research Council, Pavia, Italy. 3Present address: Institute for Molecular Health ✉ Sciences, ETHZ, Zürich, Switzerland. 4These authors contributed equally: Bruno Silva, Rajika Arora. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:3760 | https://doi.org/10.1038/s41467-021-24097-6 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-24097-6 ranscription of telomeric DNA into the long noncoding telomeric fusions24, which has been interpreted as evidence for RNA TERRA is an evolutionarily conserved feature of TERRA capping, rather than destabilizing, telomeres in ALT cells. T 1 eukaryotic cells with linear chromosomes . RNA poly- merase II produces TERRA proceeding from subtelomeric Results regions towards chromosome ends and using the C-rich telomeric Development of Transcription Activator-Like Effectors bind- strand as a template. As a result, TERRA molecules comprise ing to 29 bp repeats. To assess the short-term impact of TERRA chromosome-specific subtelomeric sequences followed by a – transcription on telomere stability in ALT cells we engineered variable number of telomeric UUAGGG repeats2 4. TERRA is Transcription Activator-Like Effectors (TALEs)34 targeting a found either dispersed throughout the nucleoplasm or associated 20 bp sequence within the 29 bp repeat consensus2 (herein with telomeric chromatin, as well as other genomic loci that – referred to as T-TALEs; Fig. 1a). Variable numbers of exact 20 bp contain or not telomeric DNA repeats4 7. The molecular sequences are found within the last 3 kb of 20 subtelomeres (3p, mechanisms mediating TERRA retention on chromosomes still 5p, 9p, 12p, 16p, 19p, 1q, 2q, 4q, 5q, 6q, 10q, 11q, 13q, 15q, 16q, need to be fully elucidated; however, the propensity of TERRA to 19q, 21q, 22q, and Xq/Yq). T-TALEs were C-terminally fused to a form RNA:DNA hybrids with its template DNA strand (telomeric – strong nuclear localization signal (NLS), four transcription R-loops or telR-loops)8 11 and the physical interaction of human repressor domains of the mSIN3 interaction domain (Enhanced TERRA with the shelterin factors TRF1 and TRF212,13 suggest Repressor Domain, SID4X), and a human influenza hemagglu- that TERRA association with telomeric DNA-containing loci tinin (HA) epitope (Fig. 1a). T-TALEs not fused to SID4X were involves RNA–DNA and RNA–protein interactions. used as controls. Transgenes were cloned downstream of a dox- The chromosomal origin of human TERRA is controversial. ycycline (dox) inducible promoter and stably integrated in U2OS Using RT-PCR and Illumina sequencing, independent labora- cells expressing the tetracycline repressor protein. Several clonal tories reported on the existence of TERRA molecules originating – cell lines were isolated in absence of dox and successively tested from a multitude of chromosome ends3,14 19. Consistently, we for dox-induced T-TALE expression by western blot and indirect previously identified CpG dinucleotide-rich tandem repeats of immunofluorescence (IF) using anti-HA antibodies. Two inde- 29 bp displaying promoter activity and located on approximately pendent cell lines for SID4X-fused T-TALEs (sid1 and sid4) and half of chromosome ends2. 29 bp repeats are positioned at vari- two for unfused T-TALEs (nls1 and nls3) were chosen for further able distances from the first telomeric repeat and their tran- experiments because: (i) transgene expression was almost unde- scriptional activity is repressed by CpG methylation2. Moreover, tectable in absence of dox; and (ii) dox treatments induced transcription factor binding sites exist on multiple subtelomeres expression of ectopic proteins homogeneously distributed across and inactivation of some of them alter TERRA levels in – the cell population, properly localized to the nucleus and at fairly cells14,20 22. However, work from the Blasco laboratory, based on similar levels in the four cell lines (Fig. 1a and Supplementary re-analysis of TERRA Illumina sequencing data and molecular Fig. 1a–c). and cell biological validation experiments, posed that human To confirm T-TALE binding specificity, we treated nls3, sid4, TERRA is mainly transcribed from one unique locus on the long and parental U2OS cells with dox for 24 h and performed arm of the chromosome 20 (20q) subtelomere23,24. The same chromatin immunoprecipitations (ChIPs) with anti-HA anti- group used CRISPR/Cas9 to delete a 8.1 kb fragment from the bodies followed by qPCR using oligonucleotide amplifying 20q subtelomere comprising 4 putative promoters in U2OS subtelomeric sequences from several chromosome ends either osteosarcoma cells and isolated several clonal lines (20q-TERRA containing or not 29 bp repeat sequences (29 bp+ and 29 bp−, KO cells). Seemingly supporting the proposed origin of TERRA, respectively). DNA in very close proximity of 29 bp repeats on 20q-TERRA KO cells displayed substantially diminished total chromosomes 9p, 10q, 15q, 16p, and Xq subtelomeres was TERRA levels when compared to parental cells23,24. enriched in nls3 and sid4 ChIP samples over parental U2OS TERRA is involved in several telomere-associated processes samples. The enrichment diminished for sequences more distant including telomerase recruitment and regulation, telomeric DNA from the 29 bp repeats on the same subtelomeres (Fig. 1b and replication, telomeric heterochromatin establishment, response to Supplementary Table 1). On the contrary, no enrichment was DNA damage at telomeres and replicative senescence initiation1. observed for DNA from 29 bp− subtelomeres (10p, 12q, 18p, Our laboratory and others have implicated TERRA also in telo- 20q, and Xp), the centromere of the X chromosome, centromeric mere elongation in telomerase-negative cancer cells with an alphoid repeats, and the beta Actin or U6 gene loci (Fig. 1b and activated Alternative Lengthening of Telomeres (ALT) Supplementary Table 1). This confirms that T-TALEs specifically mechanism8,9,24,25. ALT is a specialized pathway repairing and bind to 29 bp repeat. thus re-elongating damaged telomeres through Break-Induced Replication (BIR) occurring in the G2 and M phases of the cell cycle and requiring the DNA polymerase delta accessory subunits T-TALEs rapidly suppress TERRA transcription from 29 bp POLD3 and POLD425–29. Consistent with a function for TERRA repeat-containing chromosome ends. To test the functionality of in ALT, human ALT cells, including U2OS, are characterized by T-TALEs, we treated cells with dox for 24 h and performed RT- elevated telomeric transcription and TERRA levels and abundant qPCR to measure TERRA from 29 bp+ (9pXq, 10q, 15q, and 16p) telR-loops2,8,15. Moreover, the RNA:DNA endoribonuclease and 29 bp− (10p18p, 12q, 20q, and Xp) subtelomeres. In sid1 and RNaseH1 and the ATPase/helicase FANCM dismantle telR-loops sid4 cells, TERRA from 29 bp+ subtelomeres was greatly reduced and FANCM restricts total TERRA levels specifically in ALT cells. while TERRA from 29 bp− was not affected. In nls1 and nls3 cells, Because RNaseH1 and FANCM inactivation increase telomere no significant change in TERRA levels was observed at any instability and ALT activity, while their over-expression alleviates chromosome ends (Fig. 1c). Northern blot hybridization of total ALT8,9,30,31, we proposed that physiological damage triggered by RNA with a C-rich telomeric repeat probe did not detect sub- TERRA/telR-loops at ALT telomeres may provide the substrate stantial variations across sid and nls cells treated or not with dox for BIR-mediated telomere elongation8,9,32,33. However, due to a for 24 h (Supplementary Fig. 2). Hence, TERRA from 29 bp repeat lack of tools to rapidly suppress TERRA transcription in cells, our promoters does not majorly contribute to the cellular pool of hypothesis remained speculative. Further challenging our UUAGGG repeats. Moreover, because TERRA transcription from hypothesis, 20q-TERRA KO cells show increased telomeric 29 bp− chromosome ends is not affected